The goal of this research program is to determine how the ribosome catalyzes the evolutionarily conserved and biologically essential reaction of peptide bond formation. This has been a long term biochemical goal, yet several significant questions remain unanswered. Does the ribosome contribute chemically to catalysis? Does it utilize metal ions as catalytic cofactors? Does it utilize a general acid or a general base catalyst? If both, is the proton transfer concerted or step-wise? If neither, what other strategy is employed? How does it utilize the cis-diol at the P-site tRNA terminus to promote the reaction? What contribution is made by substrate-assisted catalysis? Outlined in the research proposal is a series of parallel, complementary, yet fundamentally different approaches that will reveal the mechanism of this biologically essential reaction. These experiments utilize a full gamut of techniques including synthetic organic chemistry, enzyme kinetics, biochemistry and structural biology. The regiospecificity, transition state chirality, the exit pathway for the growing peptidyl chain, and the identity of solvent atoms within the active site will be tested using a progressively more sophisticated series of active site inhibitors. The nature of substrate assisted catalysis and the role of metal ions in substrate activation will be tested by measuring the reaction kinetics of P-site tRNAs containing site specific chemical substitutions. The nature of the chemical transition state and the relative degree of bond order and charge on each atom involved in the reaction, will be determined by kinetic isotope effect analysis. Utilizing a series of unnatural amino acids with a broad spectrum of pKa values, the Bronsted coefficients of the peptidyl transferase reaction will be determined. This will provide further evidence for or against general base or general acid catalytic mechanisms. Enzymes function by binding more tightly to their transition states than their ground states. Determining the charge distribution of the transition state will make it possible to develop tight binding antibiotics against the ribosome and improve upon the antibiotics already utilized to treat bacterial infection. 2-3 sentence summary. The ribosome is responsible for making all the proteins in all living things. It is a primary drug target for the treatment of bacterial infection. The information gained in this research program will lead to improved antibiotics for combating disease.